Bacteria for expressing a polysaccharide depolymerase containing a novel recombinant plasmid

ABSTRACT

A segment of a bacteriophage encoding for a polysaccharide depolymerase which has been cloned and expressed in Escherichia coli is described. In particular, bacteriophage ERA103 was found to consist of five EcoRI fragments labeled: A, 7.5-kb; B, 5.0-kb; C, 2.7-kb; D, 2.1-kb and E, 1.8-kb. Fragment B encodes for the depolymerase and was cloned into the positive-selection vector pOP203(A 2   + ), pBR322 and the expression vector pKK223-3. The depolymerase is applied to rosaceous plants to prevent Fireblight caused by Erwinia amylovora by depolymerizing a polysaccharide produced by this bacteria.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to recombinant plasmids and to bacteriacontaining the recombinant plasmids which produce polysaccharidedepolymerase, wherein the recombinant plasmid contains a DNA segment ofa bacteriophage for Erwinia amylovora. In particular the presentinvention relates to Escherichia coli containing a recombinant plasmidwith a DNA segment of phage ERA103 which expresses a depolymerase.

2. Prior Art

Erwinia amylovora is recognized as the causitive agent of fireblight inrosaceous plants (Ayers, A. R., et al., Appl. Environ. Microbiol. 38:659-666 (1979)). The pathogenic strains of this bacteria have beendemonstrated to produce a polysaccharide capsule which has beenimplicated in xylem vessel occlusion and plasmolysis of xylemparenchymal cells, which are symptomatic of the disease. U.S. patentapplication Ser. No. 662,056, filed Oct. 18, 1984 now U.S. Pat. No.4,678,750 describes the use of a bacteriophage induced depolymerase fromErwinia amylovora for the treatment of this disease.

Bacteriophage encoded polysaccharide depolymerases have been describedfor many bacterial genera (Adams, M. H., et al., Virology 2: 719-736(1956) and Higashi, S., et al., J. Gen. Appl. Microbiol. 24: 143-153(1978)). Bacteriophage PEal (h) has been shown to degrade to thepolysaccharide capsule of Erwinia amylovora (Hartung, J. S., et al.,Phytopathology 72: 945 (1982)). Bacteriophage ERA103 has been found toinfect the plant pathogen E. amylovora NCPPB595 and produce adepolymerase that degrades the polysaccharide capsule of this bacteria.The problem is that it is difficult and expensive to isolate significantamounts of the depolymerase by induction with the bacteriophage.

Much more of the depolymerase might be obtained if it was possible toclone a segment of the phage DNA encoding for the depolymerase into avector plasmid. It was uncertain whether the depolymerase could beproduced without the presence of the host Erwinia amylovora or whetherthe genes responsible for encoding the depolymerase could be cloned intoa recombinant plasmid.

Objects

It is therefore an object of the present invention to provide arecombinant plasmid in a bacteria which encodes for the expression of adepolymerase. Further the present invention relates to a recombinantplasmid including a segment of a bacteriophage which infects Erwiniaamylovora and which encodes for the depolymerase. In particular it is anobject of the present invention to provide a recombinant plasmidincluding a segment of bacteriophage ERA 103 which encodes for theexpression of the depolymerase in E. coli. Finally, it is an object ofthe present invention to provide a method for producing the depolymeraseusing the recombinant plasmid which is simple and economical. These andother objects will become increasingly apparent by reference to thefollowing description and the drawings.

IN THE DRAWINGS

FIG. 1 is a restriction map of bacteriophage ERA103 DNA whereinfragments have been lettered in order of decreasing size. Dep representsthe fragment expressing the depolymerase activity.

FIG. 2 is an agarose gel electrophoresis of DNA preparations purifiedwith cesium chloride-ethidium bromide and digested with EcoRI as therestriction endonuclease enzyme. The agarose concentration was 0.7%, andmigration was from top to bottom. The contents of lanes are as follows:(A) EcoRI digested pSRQ51; (B) EcoRI digested pSRQ52; (C) EcoRI digestedpSRQ53; (D) EcoRI digested pSRQ54; (E) EcoRI digested pSRQ55; (F) EcoRIdigested bacteriophage ERA103; (G) EcoRI digested pOP203(A₂ ⁺).

GENERAL DESCRIPTION

The present invention relates to a recombinant plasmid which can expressa polysaccharide depolymerase in bacteria containing the plasmid whichcomprises: a linear segment from a bacteriophage which infects Erwiniaamylovora to produce the depolymerase and which codes for thedepolymerase; and a transformable vector plasmid segment ligated to thelinear segment to provide the recombinant plasmid.

Further the present invention relates to a bacteria which expresses apolysaccharide depolymerase because of a resident recombinant plasmidwherein the recombinant plasmid comprises: a linear segment from abacteriophage which infects Erwinia amylovora to produce thedepolymerase and which codes for the depolymerase; and a transformablevector plasmid segment ligated to the linear segment to provide therecombinant plasmid.

Finally the present invention relates to a method for producing apolysaccharide depolymerase which comprises: providing a bacteria forexpressing the polysaccharide depolymerase containing a residentrecombinant plasmid with a linear segment from a bacteriophage whichinfects Erwinia amylovora to produce the depolymerase and which codesfor the depolymerase and with a transformable vector plasmid segmentwhich has been ligated to the linear segment to provide the recombinantplasmid; growing the bacteria in a growth medium to produce thedepolymerase; and removing the depolymerase from the bacteria and growthmedium.

The preferred bacteriophage is ERA 103; however other bacteriophageswhich induce the depolymerase in Erwinia amylovora which could be usedare ERA 225 and ERA 101 which are freely available from the Universityof Nebraska, Dr. Anne K. Vidaver, Department of Plant Pathology.

The following specific description includes the mapping of thebacteriophage genome and the cloning of a fragment (B) from ERA103 into(1) pBR322 (Bolivar, et al., Methods of Enzymol 68: 245-267 (1979)), (2)the positive-selection vector pOP203(A₂ ⁺) (Winter, R. B., et al., Cell33: 877-885 (1983)), or (3) the expression vector pKK223-3 (Vievia, J.,et al., Gene 19: 259-268 (1982)), as the plasmids and expression of thedepolymerase in Escherichia coli. The positive selection vectorpOP203(A₂ ⁺) contains genes that are lethal to the bacterial host.Therefore, when DNA fragments are inserted into the unique restrictionsites, expression of genes deleterious to the host are suppressed. Theexpression vector pKK223-3 was developed for high level regulatedexpression of protein products in E. coli. This plasmid contains thestrong trp-lac (tac) promoter which facilitates the expression of clonedgenes during induction. The procedure is generally described by DeBoer,H. A., et al., P.N.A.S. 80: 21-25 (1983).

SPECIFIC DESCRIPTION

Materials and Methods

Bacterial strains and media. Bacterial strains used or constructed inthis invention are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Bacterial Strains and Plasmids                                                          Remarks          Reference                                          ______________________________________                                        Strain                                                                        Bacteria                                                                      E. coli                                                                       HB101       lacI.sup.qa        (1)                                            HB101(pOP203A.sub.2+)                                                                     contains positive selection                                                                      (1)                                                        vector                                                            HB101(pBR322)                                                                             contains plasmid pBR322                                                                          (4)                                            JM105       lacI.sup.b         (3)                                            JM105(pKK223-3)                                                                           contains expression vector                                                                       (2)                                            Plasmids                                                                      pOP203(A.sub.2.sup.+)                                                                     lac.sup.b A.sub.2.sup.c tet.sup.d                                                                (1)                                            pBR322      bla.sup.3 tet      (4)                                            pKK223-3    bla tet tac.sup.f  (4)                                            pSRQ51      phage fragment A in                                                                              present                                                    pOP203(A.sub.2.sup.+)EcoRI site                                                                  invention                                      pSRQ52      phage fragment B in                                                                              NRRL-B-                                                    pOP203(A.sub.2.sup.+) EcoRI site                                                                 15989                                          pSRQ53      phage fragment C in                                                                              present                                                    pOP203(A.sub.2.sup.+) EcoRI site                                                                 invention                                      pSRQ54      phage fragment D in                                                                              present                                                    pOP203(A.sub.2.sup.+) EcoRI site                                                                 invention                                      pSRQ55      phage fragment E in                                                                              present                                                    pOP203(A.sub.2.sup.+) EcoRI site                                                                 invention                                      pSRQ56      phage fragment B in                                                                              NRRL-B-                                                    pBR322 EcoRI site  15990                                          pSRQ57      phage fragment B in                                                                              NRRL-B-                                                    pKK223-3 EcoRI site                                                                              15991                                          pSRQ58      SphI segment of the B                                                                            present                                                    fragment in pBR322 SphI site                                                                     invention                                      ______________________________________                                         .sup.a hyperlactose repressorproducing mutation carried by F'lac              exogenate.                                                                    .sup.b lactose promoter/operator.                                             .sup.c maturation protein gene of the RNA bacteriophage Qbeta.                .sup.d tetracycline resistance.                                               .sup.e ampicillin resistance.                                                 .sup.f trp-lac promoter.                                                      (1) Winter, R. B., et al. Cell 33: 877-885 (1983).                            (2) DeBoer, H. A., et al. P.N.A.S. 80: 21-25 (1983)                           (3) Vievia, J., et al. Gene 19: 259-268 (1982).                               (4) Bolivar, F., et al., Methods Enzymol 68: 245-267 (1979).             

E. coli strains were grown in L broth (Davis, R. W., et al., A manualfor genetic engineering: advanced bacterial genetics. Cold spring HarborLaboratory, Cold Spring Harbor, N. Y. (1980)). Tetracycline (SigmaChemical Co., St. Louis, Mo.) was added to media at a concentration of10 micrograms/ml. Carbenicillin (United States Biochemical Corporation,Cleveland, OH.) was added to media at a concentration of 50micrograms/ml. Isopropyl B-D-thiogalactoside (IPTG; Sigma) was added tothe media for a final concentration of 1.0 mM where indicated.

Bacteriophage preparation

High titer stocks of bacteriophage ERA103 (10¹¹ to 10¹² PFU/ml) weredeveloped as previously described (Yamamoto, K. R., et al., Virology 40:734-744 (1970)).

Plasmid and bacteriophage DNA isolation

Plasmid DNA was isolated from E. coli by a method previously describedin the literature (Vandenbergh, P. A., et al., J. Dent. Res. 61: 497-501(1982)). Polyethylene glycol (PEG) precipitated high titer phagepreparations were centrifuged in cesium chloride-ethidium bromidegradients. The cesium chloride purified bacteriophage DNA was dialyzedovernight in 4.0 l of 0.01M tris (hydroxymethyl)aminomethane-hydrochloride (pH 8.0) containing 0.001Methylenediaminetetracetic acid. Subsequently, the bacteriophage DNApreparation was extracted twice with saturated phenol and ethanolprecipitated.

Bacterial transformations

E. coli was transformed by the CaCl₂ -heat shock method (Davis, R. W.,et al. A manual for genetic engineering: advanced bacterial genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980)), withcells harvested at an absorbance of 0.6 (600 nm).

Restriction endonuclease digestion and ligation

Restriction endonucleases and T4 DNA ligase (Bethesda ResearchLaboratories, Bethesda, MD.) were used in the buffers and under theconditions recommended by the supplier.

Construction and screening of clones

Bacteriophage ERA103 DNA was digested with EcoRI and ligated withpOP203(A₂ ⁺) or pBR322 vectors cut to completion with EcoRI. Theligation mixture contained approximately a 1:2 ratio of vector DNA tobacteriophage DNA. Bacteriophage ERA103 DNA was digested with EcoRI andligated with pKK223-3 vector cut to completion with EcoRI. The ligationmixture contained approximately a 1:10 ratio of vector DNA tobacteriophage DNA. Ligation was performed at 17° C. for 18 hours. Theligation procedure uses DNA T4 ligase as described by Bolivar et al(1979).

Determination of depolymerase activity

Bacterial cultures were grown overnight in L broth at 37° C.supplemented with either tetracycline at a concentration of 10micrograms/ml, or carbenicillin at a concentration of 50 micrograms/mldepending on the vector utilized. This culture was then deluted 1:100into 250 ml of similar media with cells harvested at an absorbance of0.9 (600 nm). All subsequent fractionation steps were performed at 4° C.The cells were then centrifuged at 8,000 xg for 30 min. The pellet wasthen suspended in sterile distilled water and centrifuged at 8,000 xgfor 30 minutes. The washed pellet was then resuspended in 10 ml of 0.01Mcitrate buffer (pH 6.0) containing 0.01M 2-mercaptoethanol. Cell-freeextracts (CFE) were prepared by passage of washed cell suspensionsthrough a French Press at 16,000 p.s.i., three times. The resultant CFEwas then centrifuged at 27,000 xg for 30 minutes to remove whole cellsand cell debris. Ammonium sulfate was added slowly to the supernatant togive a final concentration of 45% saturation and precipitated for 18hours. The ammonium sulfate precipitate was centrifuged at 27,000 xg for30 minute and dialyzed overnight against buffer. Depolymerase activitywas assayed by following the release of galactose from thepolysaccharide substrate by the method of Fairbridge et al (Fairbridge,R. A., et al., Biochem. J. 49: 423-427 (1951)). Polysaccharide wasprepared from uninfected cultures of E. amylovora NCPPB595 cultivated onsheets of cellophane overlaying tryptic soy agar, as described by Liu etal (Liu, P. V., et al., J. Infect. Dis 108: 218-228 (1961)).

One unit of enzyme is the amount of enzyme to produce 1 micromole ofgalactose equivalent per minute under standard assay conditions. Proteinconcentrations were determined by the method of Koch and Putnam (Koch,A., et al., Anal. Bicohem. 44: 239-245 (1971)).

Induction Studies

Bacterial culture were grown overnight in L broth at 37° C. supplementedwith the appropriate antibiotic. This culture was then diluted 1:100into 250 ml of similar media until the cells reached an absorbance of0.7 (600 nm). IPTG was added to a final concentration of 1 mM, andincubated until an absorbance of 0.9 (600 nm) was obtained. The cultureswere then processed as described in the previous section.

Preparation of Depolymerase

FIG. 1 which is a restriction map depicts the relative positions ofvarious restrictions endonuclease recognition sites on phage ERA103 DNAobtained using a combination of the following procedures: (i) analysisof DNA fragments obtained after digestion with two enzymes; (ii)analysis of E. coli plasmids containing phage ERA103 EcoR1 fragments.DNA from phage ERA103 was cleaved by EcoR1 into five distinct fragmentslabeled: A, 7.5-kb; B, 5.0-kb; C, 2.7-kb; D, 2.1-kb and E, 1.8-kb.Several restriction enzymes: BamHI, BstEII, ClaI, HindIII, KpnI, PstI,SalI and SstI, did not cleave the phage DNA.

The plasmid pOP203(A₂ ⁺) contains the lac promoter-operator fused to theQ beta phage maturation gene. Positive selection results when onlysurvivors with inserts in the A₂ gene grow in the presence of IPTG.Since the vector has several unique restriction sites, including EcoRI,this vector was used. Colonies were then screened for the presence ofplasmid DNA containing the five EcoRI phage fragments. All five EcoRIfragments were cloned into pOP203(A₂ ⁺) (FIG. 2). The plasmid containingstrains were grown in 250 ml of L broth containing tetracycline at 10micrograms/ml. Enzyme assays in triplicate of CFE of each clonedfragment demonstrated that enzyme activity was associated with the Bfragment which is 5.0-kb, and contained in pSRQ52. Depolymerase activitywas always associated with the supernatants of the CFE and notdemonstrated in the pellets.

Mapping data revealed the presence of SphI sites in the phage ERA103DNA. The vector pBR322 was utilized because of the insertionalinactivation of tetracycline resistance in the SphI site of this vector.Transformants that were resistant to carbenicillin and sensitive totetracycline were screened for plasmids and depolymerase activity. An E.coli strain containing the plasmid pSRQ58, a 1.5-kb portion of the Bfragment demonstrated depolymerase activity.

Additional cloning experiments utilized the expression vector pKK223-3.The B fragment was cloned in the EcoRI site of this plasmid.

Expression of the enzyme was examined utilizing various E. coli strainscontaining the cloned depolymerase fragment as shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Depolymerase activity by E. coli HB101                                        containing various plasmids.                                                                Growth                                                          Plasmid       Conditions                                                                              Enzyme sp. act..sup.a                                 ______________________________________                                        None          Uninduced O.sup.b                                               pBR322        Uninduced 0                                                     pSRQ56        Uninduced 27.1                                                  pSRQ58        Uninduced 39.0                                                  pOP203(A.sub.2.sup.+)                                                                       Uninduced 0                                                     pOP203(A.sub.2.sup.+)                                                                       Induced   0                                                     pSRQ52        Uninduced 28.0                                                  pSRQ52        Induced   34.0                                                  pKK223-3      Uninduced 0                                                     pKK223-3      Induced   0                                                     pSRQ57        Uninduced 34.0                                                  pSRQ57        Induced   47.2                                                  ______________________________________                                         .sup.a Specific activities are expressed in micromole of galactose            equivalent per minute per milligram of protein under standard assay           conditions.                                                                   .sup.b A measurement of zero implies activity of <0.05.                  

The E. coli strains containing pSRQ57 or pSRQ52 were grown and inducedwith IPTG or uninduced. The E. coli strains containing pSRQ56 or pSRQ58were also grown and compared to the above E. coli strains. The CFEsupernatants were precipitated with ammonium sulfate and dialyzedagainst buffer. All of the ammonium sulfate precipitates werestandardized to equivalent protein concentrations and assayed fordepolymerase in triplicate. The E. coli strains containing plasmids withthe tac promoter produced about twenty additional depolymerase unitsupon induction. Strains containing plasmids with only the lac promoterproduced about seven additional units when induced. Thus 13.2 and 6additional units of specific activity are shown in Table 2 for inducedversus non-induced pSRQ57 and pSRQ52, respectively. Table 2 demonstratesthat the gene coding for the depolymerase from the bacteriophage ERA103and related bacteriophages can be cloned into pBR322, pOP203(A₂ ⁺),pKK223-3 and expressed in E. coli. It can also be expressed with othervectors and bacteria as will be obvious to those skilled in the art.

The physical map of ERA103 in FIG. 1 includes the positions for cleavagesites of three restriction enzymes. Enzyme assays conducted on the cellfree extract (CFE) supernatants of the five cloned EcoRI fragments ofbacteriophage DNA demonstrated that the depolymerase activity wasassociated with the 5.0-kb B fragment. Subsequent subcloning utilizingpBR322, associated the activity within a 1.5-kb SphI fragment. When thedepolymerase activity of entire cloned B fragment in either pBR322 oruninduced pOP203(A₂ ⁺) was compared to the activity of the subclonedSphI fragment, the activity was 69% greater in the subcloned SphIfragment. Induction utilizing the entire B fragment cloned in theexpression vector pKK223-3 demonstrated a 44% increase in enzymaticactivity compared to the same fragment cloned into pBR322. Use of thepositive selection vector pOP203(A₂ ⁺) resulted in a 74% increase inactivity compared to pBR322.

The plasmid pOP203(A₂ ⁺) was derived from pOP203-3, a pMB9 plasmidcarrying the lac UV5 promoter (Fuller, F., Gene 19: 43-54 (1982)). Theexpression vector pKK223-3 contains the lacUV5 promoter and the trppromoter, i.e. tac (Amann, E., et al., Gene 25: 167-178 (1983)). Theplasmid pBR322 does not have a lac promoter. Enzymatic assays indicatedthat the expression level varied with the promoter utilized.

The polysaccharide depolymerase is a replacement for antibiotic therapyin the control of E. amylovora, the causitive agent of Fireblight.Plants were treated with the depolymerase alone or as shown in U.S.patent application Ser. No. 662,056, filed Oct. 18, 1984 now U.S. Pat.No. 4,678,750. The depolymerase can be combined with various leafwetting agents or polymeric adhesives to cause the enzyme to adhere tothe plant.

The foregoing specific description is only illustrative of the presentinvention and it is intended that the present invention be limited onlyby the hereinafter appended claims.

I claim:
 1. A recombinant plasmid which can express a polysaccharidedepolymerase in bacteria containing the plasmid which comprises:(a) alinear segment from bacteriophage ERA 103 deposited as ATCC 39824 Blwhich infects Erwinia amylovora to produce the depolymerase and whichsegment codes for the production of the depolymerase; and (b) atransformable vector plasmid segment ligated to the linear segment toprovide the recombinant plasmid wherein the recombinant plasmid isstable in the bacteria so as to express the depolymerase.
 2. Therecombinant plasmid of claim 1 as carried in E. coli NRRL-B-15989. 3.The recombinant plasmid of claim 1 as carried in E. coli NRRL-B-15990.4. The recombinant plasmid of claim 1 as carried in E. coliNRRL-B-15991.
 5. The recombinant plasmid of claim 1 wherein the linearsegment from ERA 103 is cleaved by endonuclease EcorI, SphI, or EcoI andthen SphI and the vector plasmid is selected from pOP203(A₂ +), pBR322and pKK223-3 cleaved by EcoRI and then ligated to the linear segment toprovide the recombinant plasmid.
 6. The recombinant plasmid of claim 1wherein the bacteria is E. coli.
 7. The plasmid of claim 1 wherein thevector plasmid and linear segment have been cleaved by EcoRI andligated.
 8. The plasmid of claim 1 wherein the bacteriophage lysesErwinia amylovora and wherein the polysaccharide depolymerase degrades apolysaccharide capsule around the Erwinia amylovora.
 9. The plasmid ofclaim 8 wherein the Erwinia amylovora is deposited as ATCC
 39824. 10. Abacteria which expresses a polysaccharide depolymerase because of aresident recombinant plasmid wherein the recombinant plasmidcomprises:(a) a linear segment from bacteriophage ERA 103 deposited asATCC 39824 Bl which infects Erwinia amylovora to produce thedepolymerase and which segment codes for the production of thedepolymerase; and (b) a transformable vector plasmid segment ligated tothe linear segment to provide the recombinant plasmid wherein therecombinant plasmid is stable in the bacteria so as to express thedepolymerase.
 11. The bacteria of claim 10 containing the recombinantplasmid as carried in Escherichia coli NRRL-B-15990.
 12. The bacteria ofclaim 10 containing the recombinant plasmid carried in Escherichia coliNRRL-B-15991.
 13. The bacteria of claim 10 containing the recombinantplasmid as carried in E. coli NRRL-B-15989.
 14. The bacteria of claim 10wherein the linear segment from ERA 103 is cleaved by endonucleaseEcoRI, SpHI or EcoRI and then SphI and the vector plasmid is selectedfrom pOP203 (A₂ +), pBR322 and pKK223-3 cleaved by EcoRI and thenligated to the linear segment to provide the recombinant plasmid. 15.The bacteria of claim 10 which is a strain of Escherichia coli.
 16. Thebacteria of claim 10 wherein the vector plasmid and the linear segmenthave been cleaved by EcoRI and ligated.
 17. The bacteria of claim 10wherein the bacteriophage lyses Erwinia amylovora and wherein thepolysaccharide depolymerase degrades a polysaccharide capsule around theErwinia amylovora.
 18. The bacteria of claim 17 wherein the Erwiniaamylovora is ATCC
 39824. 19. A method for producing a polysaccharidedepolymerase which comprises:(a) providing a bacteria for expressing thepolysaccharide depolymerase containing a resident recombinant plasmidwith a linear segment from bacteriophage ERA 103 deposited as ATCC 39824Bl which infects Erwinia amylovora to produce the depolymerase and whichsegment codes for the production of the depolymerase and with atransformable vector plasmid segment which has been ligated to thelinear segment to provide the recombinant plasmid wherein therecombinant plasmid is stable in the bacteria so as to express thedepolymerase; (b) growing the bacterial in a growth medium to producethe depolymerase; and (c) removing the depolymerase from the bacteriaand growth medium.
 20. The method of claim 19 wherein cells of thebacteria are ruptured and the depolymerase is removed from the rupturedcells.
 21. The method of claim 19 wherein the bacteria is Escheichiacoli.
 22. The method of claim 19 wherein the recombinant plasmid iscarried in Escherichia coli NRRL-B-15989.
 23. The method of claim 19wherein the recombinant plasmid is carried in Escherichia coliNRRL-B-15990.
 24. The method of claim 19, wherein the recombinantplasmid is carried in E. coli NRRL-B-15991.
 25. The method of claim 19wherein the linear segment from ERA 103 ATCC 39824 Bl is cleaved byendonuclease EcoRI, SphI or EcoRI and then SphI and the vector plasmidis selected from pOP203 (A₂ +), pBR322 and pKK223-3 cleaved by EcoRI andthen ligated to provide the recombinant plasmid.
 26. The method of claim19 wherein the bacteriophage lyses Erwinia amylovora and wherein thedepolymerase degrades a polysaccharide capsule of the Erwinia amylovora.27. The method of claim 26 wherein the Erwinia amylovora is ATCC 39824.